Transplantation of adipose-derived mesenchymal stem cell sheets directly into the kidney suppresses the progression of renal injury in a diabetic nephropathy rat model

Abstract

Aims/introduction: Adipose-derived mesenchymal stem cell (ASC) transplantation is a promising therapy for diabetic nephropathy (DN). However, intravascular administration of ASCs is associated with low engraftment in target organs. Therefore, we considered applying the cell sheet technology to ASCs. In this study, ASC sheets were directly transplanted into the kidneys of a DN rat model, and therapeutic consequences were analyzed.

Materials and methods: Adipose-derived mesenchymal stem cells were isolated from adipose tissues of 7-week-old enhanced green fluorescent protein rats, and ASC sheets were prepared using a temperature-responsive culture dish. A DN rat model was established from 5-week-old Spontaneously Diabetic Torii fatty rats. Seven-week-old DN rats (n = 21) were assigned to one of the following groups: sham-operated (n = 6); ASC suspension (6.0 × 10⁶ cells/mL) administered intravenously (n = 7); six ASC sheets transplanted directly into the kidney (n = 8). The therapeutic effect of the cell sheets was determined based on urinary biomarker expression and histological analyses.

Results: The ASC sheets survived under the kidney capsule of the DN rat model for 14 days after transplantation. Furthermore, albuminuria and urinary tumor necrosis factor-α levels were significantly lower in the ASC sheets transplanted directly into the kidney group than in the sham-operated and ASC suspension administered intravenously groups (P < 0.05). Histologically, the ASC sheets transplanted directly into the kidney group presented mild atrophy of the proximal tubule and maintained the renal tubular structure.

Conclusions: Transplantation of ASC sheets directly into the kidney improved transplantation efficiency and suppressed renal injury progression. Therefore, the ASC sheet technology might be a promising novel treatment for DN.

Keywords: Adipose-derived mesenchymal stem cell; Cell sheet; Renal injury.

Figure 1

Schematic representation of the experimental procedure for transplanting adipose‐derived mesenchymal stem cell (ASC) sheets into diabetic nephropathy rats. (a) ASCs were isolated from epididymal adipose tissue of enhanced green fluorescent protein rats (scale bar, 30 μm). Macro image of fabricated ASC sheet: (b) bright field and(c) dark field. (d) The ASC sheets were directly transplanted into the kidney of a diabetic nephropathy rat model. (e) Macro image of the kidneys immediately after transplantation. Diabetic nephropathy rats (aged 7‐weeks‐old) were randomly divided into three groups. Urine samples were collected on 0, 7 and 14 days after transplantation, and rats were killed 14 days after transplantation. (f) Red line: after transplantation.

Figure 2

Characteristics of adipose‐derived mesenchymal stem cell (ASCs). ASCs were confirmed on the basis of colony formation, adipogenesis and osteogenesis. (a) They were stained with crystal violet to confirm colony formation. (b) The fourth passage ASCs were stained with Oil Red O to detect adipogenesis (scale bar, 50 μm). (c) They were stained with alizarin red S to detect osteogenesis (scale bar, 50 μm). (d–h) Cell surface markers of mesenchymal stem cells were evaluated using flow cytometry. The surfaces of ASCs were positive for CD29 and CD90 and negative for CD11b, CD31 and CD45

Figure 3

Adipose‐derived mesenchymal stem cell (ASC) sheets directly transplanted into the kidney survived for 14 days. Macro image of the kidney 14 days after transplantation: (a) bright field and (b) dark field. Immunohistochemical image of the kidney 14 days after transplantation: (c) ASC i.v. group and (d) ASC sheet group (scale bar, 50 μm). GFP, green fluorescent protein.

Figure 4

Adipose‐derived mesenchymal stem cell (ASC) sheets suppressed glomerular injury. (a,b) Change in albuminuria and proteinuria after transplantation. White circles, sham group; black triangles, ASC i.v. group; white squares, ASC sheet group. (c) Urinary podocalyxin levels 14 days after transplantation. Data are presented as the mean ± standard error of the mean (n = 6–8/group); *P < 0.05.

Figure 5

Adipose‐derived mesenchymal stem cell (ASC) sheets suppressed renal tubular injury. The renal cortex of each group, 14 days after transplantation, was stained with the periodic acid/Schiff base. Representative images are shown. (a–c) Low magnification (scale bar, 500 μm). (d–f) High magnification (scale bar, 50 μm). (g,h) Levels of urinary L‐FABP and KIM‐1 14 days after transplantation. Data are presented as the mean ± standard error of the mean (n = 6–8/group); *P < 0.05.

Figure 6

Adipose‐derived mesenchymal stem cell (ASC) sheets suppressed chronic inflammation in the kidney. (a,b) Levels of urinary tumor necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6) 14 days after transplantation. Data are presented as mean ± standard error of the mean (n = 6–8/group); *P < 0.05.